Novel mechanisms of thrombo-inflammation during infection: spotlight on neutrophil extracellular trap-mediated platelet activation.

A state-of-the-art lecture titled "novel mechanisms of thrombo-inflammation during infection" was presented at the ISTH Congress in 2022. Platelet, neutrophil, and endothelial cell activation coordinate the development, progression, and resolution of thrombo-inflammatory events during infection. Activated platelets and neutrophil extracellular traps (NETs) are frequently observed in patients with sepsis and COVID-19, and high levels of NET-derived damage-associated molecular patterns (DAMPs) correlate with thrombotic complications. NET-associated DAMPs induce direct and indirect platelet activation, which in return potentiates neutrophil activation and NET formation. These coordinated interactions involve multiple receptors and signaling pathways contributing to vascular and organ damage exacerbating disease severity. This state-of-the-art review describes the main mechanisms by which platelets support NETosis and the key mechanisms by which NET-derived DAMPs trigger platelet activation and the formation of procoagulant platelets leading to thrombosis. We report how these DAMPs act through multiple receptors and signaling pathways differentially regulating cell activation and disease outcome, focusing on histones and S100A8/A9 and their contribution to the pathogenesis of sepsis and COVID-19. We further discuss the complexity of platelet activation during NETosis and the potential benefit of targeting selective or multiple NET-associated DAMPs to limit thrombo-inflammation during infection. Finally, we summarize relevant new data on this topic presented during the 2022 ISTH Congress.

Role of lymphoid lineage cells aberrantly expressing alarmins S100A8/A9 in determining the severity of COVID-19.

BACKGROUND: Alarmins S100A8 and S100A9 are recognized as hallmarks of severe COVID-19 and are primarily produced in myeloid cells, such as monocytes and neutrophils. As single-cell RNA-sequencing (scRNA-seq) data from patients with COVID-19 revealed the expression of S100A8/A9 in lymphoid cells in patients with severe COVID-19. OBJECTIVE: We investigated the characteristics of lymphoid cells expressing S100A8/A9 in COVID-19 patients. METHODS: Publicly available scRNA-seq data from patients with mild (N = 12) or severe (N = 7) COVID-19 were reanalyzed. The data were further divided into the following two groups based on the time of sample collection (from infection-onset): within 6 days (early phase) and after 6 days (late phase). Differential expression and gene set enrichment analyses were performed between S100A8/A9High and S100A8/A9Low lymphoid cells. Finally, cell-cell interaction analysis was performed to investigate the role of lymphoid cells expressing high levels of S100A8/A9 in COVID-19. RESULTS: S100A8/A9 overexpression was observed in lymphoid cells, including B cells, T cells, and NK cells, in patients with severe COVID-19 (compared to patients with mild COVID-19). Cells exhibiting strong interferon/cytokine responses were found to be associated with the severity of COVID-19. Furthermore, differences in S100A8/A9-TLR4/RAGE interactions were confirmed between patients with severe and mild disease. CONCLUSIONS: Lymphoid cells overexpressing S100A8/A9 contribute to the dysregulation of the innate immune response in patients with severe COVID-19, specifically during the early phase of infection. This study fosters a better understanding of the hyper-induction of pro-inflammatory cytokine expression and the generation of a cytokine storm in response to COVID-19 infection.

RAGE pathway activation and function in chronic kidney disease and COVID-19.

The multi-ligand receptor for advanced glycation end-products (RAGE) and its ligands are contributing factors in autoimmunity, cancers, and infectious disease. RAGE activation is increased in chronic kidney disease (CKD) and coronavirus disease 2019 (COVID-19). CKD may increase the risk of COVID-19 severity and may also develop in the form of long COVID. RAGE is expressed in essentially all kidney cell types. Increased production of RAGE isoforms and RAGE ligands during CKD and COVID-19 promotes RAGE activity. The downstream effects include cellular dysfunction, tissue injury, fibrosis, and inflammation, which in turn contribute to a decline in kidney function, hypertension, thrombotic disorders, and cognitive impairment. In this review, we discuss the forms and mechanisms of RAGE and RAGE ligands in the kidney and COVID-19. Because various small molecules antagonize RAGE activity in animal models, targeting RAGE, its co-receptors, or its ligands may offer novel therapeutic approaches to slowing or halting progressive kidney disease, for which current therapies are often inadequate.

Calprotectin Levels and Neutrophil Count Are Prognostic Markers of Mortality in COVID-19 Patients.

Inflammation plays a crucial role in worsening coronavirus disease (COVID-19). Calprotectin is a pro-inflammatory molecule produced by monocytes and neutrophilic granulocytes. The aim of the study was to evaluate both the prognostic role of circulating calprotectin levels and neutrophil count toward fatal outcome in COVID-19 patients. We retrospectively collected and analyzed data on 195 COVID-19 adult patients, 156 hospitalized in the infectious disease unit and 39 in the intensive care unit (ICU). Calprotectin levels and neutrophil counts measured at the first hospitalization day were higher in the patients with a fatal outcome than in surviving ones. The association of high calprotectin levels and neutrophil count to patient death remain significant by logistic regression, independent of patient age. ROC curves analysis for calprotectin levels and neutrophil count revealed a good discriminatory power toward survival (area under the curve of 0.759 and 0.843, respectively) and identified the best cut-off (1.66 mg/L and 16.39 × 103/µL, respectively). Kaplan-Meier analysis confirmed the prognostic role of high calprotectin levels and neutrophil count in death prediction. In conclusion, this study highlights that calprotectin levels together with neutrophil count should be considered as biomarkers of mortality in COVID-19 patients.

Circulating Calprotectin as a Predictive and Severity Biomarker in Patients with COVID-19.

BACKGROUND: New tools for the assessment and prediction of the severity of hospitalized COVID-19 patients can help direct limited resources to patients with the greatest need. Circulating levels of calprotectin (S100A8/S100A9) reflect inflammatory activity in multiple conditions, and have been described as being elevated in COVID-19 patients, but their measurement is not routinely utilized. The aim of our study was to assess the practical and predictive value of measuring circulating calprotectin levels in patients at admission and during their hospitalization. METHODS: Circulating calprotectin levels were measured in 157 hospitalized patients with COVID-19 using an automated quantitative chemiluminescent assay. RESULTS: Circulating calprotectin levels were strongly correlated with changing respiratory supplementation needs of patients. The overall trajectory of circulating calprotectin levels generally correlated with patient improvement or deterioration. CONCLUSIONS: Routine measurement of circulating calprotectin levels may offer a valuable tool to assess and monitor hospitalized patients with COVID-19, as well as other acute inflammatory conditions.

Dynamics of circulating calprotectin accurately predict the outcome of moderate COVID-19 patients.

BACKGROUND: Severe COVID-19 is associated with a high circulating level of calprotectin, the S100A8/S100A9 alarmin heterodimer. Baseline calprotectin amount measured in peripheral blood at diagnosis correlates with disease severity. The optimal use of this biomarker along COVID-19 course remains to be delineated. METHODS: We focused on patients with a WHO-defined moderate COVID-19 requiring hospitalization in a medical ward. We collected plasma and serum from three independent cohorts (N = 626 patients) and measured calprotectin amount at admission. We performed longitudinal measures of calprotectin in 457 of these patients (1461 samples) and used a joint latent class mixture model in which classes were defined by age, body mass index and comorbidities to identify calprotectin trajectories predicting the risk of transfer into an intensive care unit or death. FINDINGS: After adjustment for age, sex, body mass index and comorbidities, the predictive value of baseline calprotectin in patients with moderate COVID19 could be refined by serial monitoring of the biomarker. We discriminated three calprotectin trajectories associated with low, moderate, and high risk of poor outcome, and we designed an algorithm available as online software (https://calpla.gustaveroussy.fr:8443/) to monitor the probability of a poor outcome in individual patients with moderate COVID-19. INTERPRETATION: These results emphasize the clinical interest of serial monitoring of calprotectin amount in the peripheral blood to anticipate the risk of poor outcomes in patients with moderate COVID-19 hospitalized in a standard care unit. FUNDING: The study received support (research grants) from ThermoFisher immunodiagnostics (France) and Gustave Roussy Foundation.

Short Communication: S100A8 and S100A9, Biomarkers of SARS-CoV-2 Infection and Other Diseases, Suppress HIV Replication in Primary Macrophages.

S100A8 and S100A9 are members of the Alarmin family; these proteins are abundantly expressed in neutrophils, form a heterodimer complex, and are secreted in plasma on pathogen infection or acute inflammatory diseases. Recently, both proteins were identified as novel biomarkers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and were shown to play key roles in inducing an aggressive inflammatory response by mediating the release of large amounts of pro-inflammatory cytokines, called the "cytokine storm." Although co-infection with SARS-CoV-2 in people living with HIV-1 may result in an immunocompromised status, the role of the S100A8/A9 complex in HIV-1 replication in primary T cells and macrophages is still unclear. Here, we evaluated the roles of the proteins in HIV replication to elucidate their functions. We found that the complex had no impact on virus replication in both cell types; however, the subunits of S100A8 and S100A9 inhibit HIV in macrophages. These findings provide important insights into the regulation of HIV viral loads during SARS-CoV-2 co-infection.

Calprotectin and Imbalances between Acute-Phase Mediators Are Associated with Critical Illness in COVID-19.

The trajectory from moderate and severe COVID-19 into acute respiratory distress syndrome (ARDS) necessitating mechanical ventilation (MV) is a field of active research. We determined serum levels within 24 h of presentation of 20 different sets of mediators (calprotectin, pro- and anti-inflammatory cytokines, interferons) of patients with COVID-19 at different stages of severity (asymptomatic, moderate, severe and ARDS/MV). The primary endpoint was to define associations with critical illness, and the secondary endpoint was to identify the pathways associated with mortality. Results were validated in serial measurements of mediators among participants of the SAVE-MORE trial. Levels of the proinflammatory interleukin (IL)-8, IL-18, matrix metalloproteinase-9, platelet-derived growth factor (PDGF)-B and calprotectin (S100A8/A9) were significantly higher in patients with ARDS and MV. Levels of the anti-inflammatory IL-1ra and IL-33r were also increased; IL-38 was increased only in asymptomatic patients but significantly decreased in the more severe cases. Multivariate ordinal regression showed that pathways of IL-6, IL-33 and calprotectin were associated with significant probability for worse outcome. Calprotectin was serially increased from baseline among patients who progressed to ARDS and MV. Further research is needed to decipher the significance of these findings compared to other acute-phase reactants, such as C-reactive protein (CRP) or ferritin, for the prognosis and development of effective treatments.

Elevated Levels of Neutrophil Activated Proteins, Alpha-Defensins (DEFA1), Calprotectin (S100A8/A9) and Myeloperoxidase (MPO) Are Associated With Disease Severity in COVID-19 Patients.

Understanding of the basis for severity and fatal outcome of SARS-CoV-2 infection is of paramount importance for developing therapeutic options and identification of prognostic markers. So far, accumulation of neutrophils and increased levels of pro-inflammatory cytokines are associated with disease severity in COVID-19 patients. In this study, we aimed to compare circulatory levels of neutrophil secretory proteins, alpha-defensins (DEFA1), calprotectin (S100A8/A9), and myeloperoxidase (MPO) in COVID-19 patients with different clinical presentations. We studied 19 healthy subjects, 63 COVID-19 patients with mild (n=32) and severe (n=31) disease, 23 asymptomatic individuals identified through contact tracing programme and 23 recovering patients (1-4 months post-disease). At the time of disease presentation, serum levels of DEFA1 were significantly higher in patients with mild (mean230 ± 17, p<0.0001) and severe (mean452 ± 46, p<0.0001) disease respectively in comparison to healthy subjects (mean113 ± 11). S100A8/A9 proteins were significantly higher in COVID-19 patients (p<0.0001) irrespective of disease severity. The levels of DEFA1, S100A8/A9 and MPO reduced to normal in recovering patients and comparable to healthy subjects. Surprisingly, DEFA1 levels were higher in severe than mild patients in first week of onset of disease (p=0.004). Odds-ratio analysis showed that DEFA1 could act as potential biomarker in predicting disease severity (OR=11.34). In addition, levels of DEFA1 and S100A8/A9 were significantly higher in patients with fatal outcome (p=0.004 and p=0.03) respectively. The rise in DEFA1 levels was independent of secondary infections. In conclusion, our data suggest that induction of elevated levels of alpha-defensins and S100A8/A9 is associated with poor disease outcome in COVID-19 patients.

S100A8/A9 in COVID-19 pathogenesis: Impact on clinical outcomes.

Coronavirus disease 2019 (COVID-19) has a broad range of clinical manifestations, highlighting the need for specific diagnostic tools to predict disease severity and improve patient prognosis. Recently, calprotectin (S100A8/A9) has been proposed as a potential biomarker for COVID-19, as elevated serum S100A8/A9 levels are associated with critical COVID-19 cases and can distinguish between mild and severe disease states. S100A8/A9 is an alarmin that mediates host proinflammatory responses during infection and it has been postulated that S100A8/A9 modulates the cytokine storm; the hallmark of fatal COVID-19 cases. However, it has yet to be determined if S100A8/A9 is a bona-fide biomarker for COVID-19. S100A8/A9 is widely implicated in a variety of inflammatory conditions, such as cystic fibrosis (CF) and chronic obstructive pulmonary disorder (COPD), as well as pulmonary infectious diseases, including tuberculosis and influenza. Therefore, understanding how S100A8/A9 levels correlate with immune responses during inflammatory diseases is necessary to evaluate its candidacy as a potential COVID-19 biomarker. This review will outline the protective and detrimental roles of S100A8/A9 during infection, summarize the recent findings detailing the contributions of S100A8/A9 to COVID-19 pathogenesis, and highlight its potential as diagnostic biomarker and a therapeutic target for pulmonary infectious diseases, including COVID-19.

S100A8 may govern hyper-inflammation in severe COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic threatens human species with mortality rate of roughly 2%. We can hardly predict the time of herd immunity against and end of COVID-19 with or without success of vaccine. One way to overcome the situation is to define what delineates disease severity and serves as a molecular target. The most successful analogy is found in BCR-ABL in chronic myeloid leukemia, which is the golden biomarker, and simultaneously, the most effective molecular target. We hypothesize that S100 calcium-binding protein A8 (S100A8) is one such molecule. The underlying evidence includes accumulating clinical information that S100A8 is upregulated in severe forms of COVID-19, pathological similarities of the affected lungs between COVID-19 and S100A8-induced acute respiratory distress syndrome (ARDS) model, homeostatic inflammation theory in which S100A8 is an endogenous ligand for endotoxin sensor Toll-like receptor 4/Myeloid differentiation protein-2 (TLR4/MD-2) and mediates hyper-inflammation even after elimination of endotoxin-producing extrinsic pathogens, analogous findings between COVID-19-associated ARDS and pre-metastatic lungs such as S100A8 upregulation, pulmonary recruitment of myeloid cells, increased vascular permeability, and activation coagulation cascade. A successful treatment in an animal COVID-19 model is given with a reagent capable of abrogating interaction between S100A8/S100A9 and TLR4. In this paper, we try to verify our hypothesis that S100A8 governs COVID-19-associated ARDS.

Upregulation of oxidative stress gene markers during SARS-COV-2 viral infection.

Severe viral infections, including SARS-COV-2, could trigger disruption of the balance between pro-oxidant and antioxidant mediators; the magnitude of which could reflect the severity of infection and lung injury. Using publicly available COVID-19 transcriptomic datasets, we conducted an in-silico analyses to evaluate the expression levels of 125 oxidative stress genes, including 37 pro-oxidant genes, 32 oxidative-responsive genes, and 56 antioxidant genes. Seven oxidative stress genes were found to be upregulated in whole blood and lung autopsies (MPO, S100A8, S100A9, SRXN1, GCLM, SESN2, and TXN); these genes were higher in severe versus non-severe COVID-19 leucocytes. Oxidative genes were upregulated in inflammatory cells comprising macrophages and CD8+ T cells isolated from bronchioalveolar fluid (BALF), and neutrophils isolated from peripheral blood. MPO, S100A8, and S100A9 were top most upregulated oxidative markers within COVID-19's lung autopsies, whole blood, leucocytes, BALF derived macrophages and circulating neutrophils. The calprotectin's, S100A8 and S100A9 were upregulated in SARS-COV-2 infected human lung epithelium. To validate our in-silico analysis, we conducted qRT-PCR to measure MPO and calprotectin's levels in blood and saliva samples. Relative to uninfected donor controls, MPO, S100A8 and S100A9 were significantly higher in blood and saliva of severe versus asymptomatic COVID-19 patients. Compared to other different viral respiratory infections, coronavirus infection showed a prominent upregulation in oxidative stress genes with MPO and calprotectin at the top of the list. In conclusion, SARS-COV-2 induce the expression of oxidative stress genes via both immune as well as lung structural cells. The observed correlation between oxidative stress genes dysregulation and COVID-19 disease severity deserve more attention. Mechanistical studies are required to confirm the correlation between oxidative stress gene dysregulation, COVID-19 severity, and the net oxidative stress balance.

Circulating Calprotectin as a Biomarker of COVID-19 Severity.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although demographic and clinical parameters such as sex, age, comorbidities, genetic background and various biomarkers have been identified as risk factors, there is an unmet need to predict the risk and onset of severe inflammatory disease leading to poor clinical outcomes. In addition, very few mechanistic biomarkers are available to inform targeted treatment of severe (auto)-inflammatory conditions associated with COVID-19. Calprotectin, also known as S100A8/S100A9, MRP8/14 (Myeloid-Related Protein) or L1, is a heterodimer involved in neutrophil-related inflammatory processes. In COVID-19 patients, calprotectin levels were reported to be associated with poor clinical outcomes such as significantly reduced survival time, especially in patients with severe pulmonary disease. AREAS COVERED: Pubmed was searched using the following keywords: Calprotectin + COVID19, S100A8/A9 + COVID19, S100A8 + COVID-19, S100A9 + COVID-19, MRP8/14 + COVID19; L1 + COVID-19 between May 2020 and 8 March 2021. The results summarized in this review provide supporting evidence and propose future directions that define calprotectin as an important biomarker in COVID-19. EXPERT OPINION: Calprotectin represents a promising serological biomarker for the risk assessment of COVID-19 patients.

Induction of alarmin S100A8/A9 mediates activation of aberrant neutrophils in the pathogenesis of COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic poses an unprecedented public health crisis. Evidence suggests that SARS-CoV-2 infection causes dysregulation of the immune system. However, the unique signature of early immune responses remains elusive. We characterized the transcriptome of rhesus macaques and mice infected with SARS-CoV-2. Alarmin S100A8 was robustly induced in SARS-CoV-2-infected animal models as well as in COVID-19 patients. Paquinimod, a specific inhibitor of S100A8/A9, could rescue the pneumonia with substantial reduction of viral loads in SARS-CoV-2-infected mice. Remarkably, Paquinimod treatment resulted in almost 100% survival in a lethal model of mouse coronavirus infection using the mouse hepatitis virus (MHV). A group of neutrophils that contributes to the uncontrolled pathological damage and onset of COVID-19 was dramatically induced by coronavirus infection. Paquinimod treatment could reduce these neutrophils and regain anti-viral responses, unveiling key roles of S100A8/A9 and aberrant neutrophils in the pathogenesis of COVID-19, highlighting new opportunities for therapeutic intervention.

Elevated Calprotectin and Abnormal Myeloid Cell Subsets Discriminate Severe from Mild COVID-19.

Blood myeloid cells are known to be dysregulated in coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2. It is unknown whether the innate myeloid response differs with disease severity and whether markers of innate immunity discriminate high-risk patients. Thus, we performed high-dimensional flow cytometry and single-cell RNA sequencing of COVID-19 patient peripheral blood cells and detected disappearance of non-classical CD14LowCD16High monocytes, accumulation of HLA-DRLow classical monocytes (Human Leukocyte Antigen - DR isotype), and release of massive amounts of calprotectin (S100A8/S100A9) in severe cases. Immature CD10LowCD101-CXCR4+/- neutrophils with an immunosuppressive profile accumulated in the blood and lungs, suggesting emergency myelopoiesis. Finally, we show that calprotectin plasma level and a routine flow cytometry assay detecting decreased frequencies of non-classical monocytes could discriminate patients who develop a severe form of COVID-19, suggesting a predictive value that deserves prospective evaluation.